Hypoxic pulmonary vasoconstriction (HPV) serves as an important regulatory mechanism to maintain adequate arterial oxygenation in response to hypoxia, but can also result in pulmonary hypertension. Increasing evidence indicates that a rise in intracellular Ca2+ concentration ([Ca2+]i) in pulmonary artery smooth muscle cells (PASMCs) plays a crucial role in the development of HPV. The hypoxic rise in [Ca2+]i can occur due to Ca2+ release from the sarcoplasmic reticulum (SR) and extracellular Ca2+ influx following inhibition of voltage-dependent K+ channels and activation of store-operated Ca2+ channels. The inhibition of voltage-dependent K+ channels and activation of store-operated channels by hypoxia are possibly associated with the SR Ca2+ release. The cellular and molecular processes coupling hypoxia to Ca2+ release, however, are incompletely understood. Our recent findings, together with previous publications, suggest that mitochondrial electron transport chain may function as a primary hypoxia sensor by increasing the generation of reactive oxygen species (ROS), which activate phospholipase C (PLC) and protein kinase C (PKC). Both ROS and PKC are likely to produce a synergetic effect on Ca2+ release channels. In addition, PKC may stimulate NADPH oxidase and then generate more ROS, providing a positive feedback mechanism to mediate hypoxic responses. To test this hypothesis, this application will address the following questions (specific aims): 1) Is the PLC-PKC-NADPH oxidase signaling involved in hypoxic Ca2+ release? 2) Is the PLC-PKC-NADPH oxidase signaling activated following the excessive generation of mitochondrial ROS during hypoxic stimulation? and 3) How do ROS produced by hypoxia activate ryanodine receptors to cause Ca2+ release in PASMCs. These aims will be pursued by measuring hypoxic ROS generation and Ca2+ release, mRNA and protein expression of individual PLC and PKC isoforms, as well as NADPH oxidase subunits, and the activity of individual PLC and PKC isoforms, as well as NADPH oxidase in mouse PASMCs. Pharmacological inhibitors, small interfering RNAs and transgenic mice will also be used to define the coupling of hypoxia to Ca2+ release. The findings from this proposal will enhance our understanding of cellular molecular mechanisms for hypoxic [Ca2+]i rise in PASMCs and associated HPV, and may lead to identify potential novel targets to treat pulmonary hypertension.